Project Summary Coherent imaging technologies including as laser scanning confocal, multi-photon excited fluorescence (MPEF), second-harmonic generation (SHG), and coherent Anti-Stokes Raman scattering (CARS) microscopy have become powerful tools for biological research. However, the spatial heterogeneity of biological tissues presents a source of optical scattering that severely compromises the ability of these methods to acquire undistorted, high-resolution images at depths larger than a few hundred micrometers. Recently, investigators have sought to use an approach known as adaptive optics to mitigate scattering and optical system aberrations in microscopy. Adaptive optics represents an empirical approach to improve image quality by modifying the excitation wave front characteristics in order to improve the detected signal. This method has enabled some investigators to retrieve information at tissue depths that were previously inaccessible. However, the current implementations of adaptive optics, based on experimentally accessible parameters, do not ensure that the modification of the excitation signal properly corrects for the scattering- induced aberrations produced by tissue scattering. However it has been shown that adaptive optics optimization, using parameters measured from the detected optical signal, does not ensure that the modified excitation wave front characteristics properly corrected for the aberrations produced by the sample. We believe that a general and robust strategy to manage the depth and resolution limitations in optical microscopy must go beyond empirical optimization and be based on a mechanistic understanding of the light scattering. In this proposal, we propose to develop a fundamental ?bottom-up? approach to adaptive optics that utilizes a mechanistic treatment of scattering mechanisms in tissue that models how optical signal aberrations arise and propagate in optical microscopy. Using our recently developed Huygens? Fresnel Wave-based Electric Field Superposition (HF-WEFS) method that efficiently describes focused beam propagation in scattering media, we will mathematically model the effect of adaptive optics in two photon excited fluorescence (TPEF), SHG and CARS microscopy systems. We will apply these models to predict the effects of adaptive optics correction strategies when applied to the imaging of scattering media in above microscope techniques. We will verify our model predictions through experimental measurements. This project is expected to provide novel insights into an unexplored area of microscopy. We expect that the results of this investigation will not only fuel a better understanding of this process, but also will drive innovations to improve the capabilities in coherent optical imaging microscopy. This in turn will make possible improvements in the imaging of structural and functional information in thick tissues for use in studies spanning the fields of neurobiology, embryology, cell biology, developmental biology, cancer biology, and dermatology.